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Protocol no. 32
DUST TOXICITY IN RAT ALVEOLAR MACROPHAGE CULTURES

Macrophage cells in culture may be exposed to particulate matter, and resultant effects upon cell viability determined by vital dye exclusion and enzyme leakage assays.

CONTACT

Prof. Dr. Yrjö Collan
Turun Yliopisto Klinis-Teoreettinen Laitos Patologian OsastoKiinamyllynkatu 10 SF-20520 Turku NG1 1NB Finland Tel: Finland - 21-6337317, 21-63371 Fax: Finland - 21-331126

RATIONALE

Many compounds, when inhaled as dust particles, have been found to be toxic to the respiratory system with long-term exposure resulting in the development of pneumoconiotic fibrotising lung disease. One of the initiating factors in fibrogenic lung disease is believed to be the direct damage inflicted upon the alveolar macrophages, hence these cells may represent a suitable in vitro screening system to investigate whether particulate matter is likely to be harmful when inhaled over an extended period. Macrophages can be isolated, cultured and exposed to suspensions of particulate matter. Damage may then be assessed in two ways, the first is simply by estimating cell death. The second parameter, i.e. lactate dehydrogenase (LDH) activity in the supernatant, provides a means of determining cell membrane damage as indicated by leakage of the enzyme, LDH, out of the cells into the medium. The damage to the cells as a result of exposure can then be used to assess whether inhalation of the dust may be harmful in vivo.

BASIC PROCEDURE

Remove the lungs from anaesthetised animals and lavage. Centrifuge the alveolar macrophages thus obtained and suspend in culture medium. Estimate cell number, dilute to 1 x 106 cells/ml, and plate out. Once the cells have adhered rinse the cultures and add fresh medium. After 24 hour incubation, the cells should be exposed to medium containing particulate matter for a further 24 hour period. After this time the medium and harvested cells should be collected and the percentage cell death established (using a Trypan blue staining technique). The cells should then be centrifuged and an aliquot of the supernatant (culture medium) be removed for assessment of LDH activity.

CRITICAL ASSESSMENT

This test system provides a rapid, sensitive and relatively inexpensive means of assessing the harmful effects of dust particles. In vitro haemolysis, an acute toxicity test, has been used in the past to detect the potential toxicity of particulate matter. However, as alveolar macrophages appear to be one of the primary sites of damage in the initial stages of fibrogenic lung disease, they are probably a more suitable cell to be used in an in vitro screening system and are likely to reflect the in vivo situation more closely. A number of enzyme activities have been examined by the author, including aspartate amino transferase, acid phosphatase and alanine amino transferase activity. Although a certain degree of reproducibility and correlation to cell damage were found, LDH activity was the most sensitive and readily detectable of the enzyme activities examined. Determination of this enzyme activity is, therefore, the preferred indicator of cell damage. Care should be taken to treat serum used to supplement culture medium so that any lactate dehydrogenase activity is removed. It should also be noted that the serum may, to some extent, exert an inhibitory effect upon the enzyme assay, although the incorporation of appropriate controls in the experimental protocol will allow for this eventuality. There are three possible methods of examining the cells after exposure to potential toxins: 1) The medium of the cultures with the naturally detached floating macrophages is collected and analysed. 2) The medium of the cultures is removed, fresh medium added, macrophages detached from the bottom of the well, then new medium with the harvested cells collected and analysed. 3) The macrophages are detached from the bottom of the well without adding fresh medium; these are collected and analysed. The third option has proved to be the most effective and consistent in identifying cell damage and best correlates to the occurrence of cell death. Modifications of this test system which use shorter incubation times are currently being developed by colleagues of the author.

TEST STATUS

In-house use

REFERENCES

  1. Committee on Enzymes of the Scandinavian Society for Clinical Chemistry and Clinical Physiology (1974) Recommended methods for the determination of four enzymes in blood. Scand. J. Clin. Lab. Invest., 33, 290-306.
  2. Pasanen, J.T. (1982) Alveolaariset ja peritoneaaliset makrofagit pölyjen sytotoksisuuden testauksessa in vitro. Pro gradu-tutkielma. Jyväskylän yliopisto. Moniste: Työterveyslaitos, Helsinki.
  3. Collan, Y., Kosma, V-M., Kalliokoski, P., Seppä, A., Kulju, T., Väänänen, I., Remola-Pärssinen, E., Miettinen, R., Pietilä, L., Gidlund-Marjanen, A.-L., Manninen, R., Anttonen, H., Tossavainen, A., Husman, K., Huuskonen, M.S., Rytkönen, E., Lehtinen, A., Kauppinen, H., Mikkonen, A., Koistinen, S., Karjalainen, T. & Härmälä, O. (1985) Siilinjärven apatiittiesiintymän richteriitti: biologinen vaikutus ja työhygieeninen merkitys. Kuopio.
  4. Collan, Y., Kosma V.-M., Anttonen, H. & Kulju, T. (1986) Toxicity of richterite in hemolysis test and macrophage cultures. In: Toxic interfaces of neurones, smoke and genes. Arch. Toxicol. Suppl., 9, 292-293.
  5. Holopainen, M., Collan, Y., Kosma V.-M, Kalliokoski, P., Kulju, T., Anttonen, H., Tossavainen, A. & Kauppinen, H. Evidence for toxicity of phlogopite in hemolysis and macrophage tests. In: Proceedings of the 2nd International Symposium on Occupational Health and Safety in Mining and Tunneling, Prague, Sept. 23-26, 1986, pp 65-72.
  6. Collan, Y., Kosma, V-M., Kulju, T., Väänänen, I., Remola-Pärssinen, E., Pesonen, E., Puhakainen, R., Rytöluoto-Kärkkäinen, R., Manninen, R. & Pasanen, J. Estimation of Dust Toxicity in Rat Alveolar Macrophage Cultures. In: Safety Evaluation of Chemicals on Laboratory Animals, Proceedings of the Finnish-Soviet Symposium, Kuopio 20-22 May 1986, (eds. Nevalainen, T., Voipio, H.-M. & Haataja, H. (eds.). Kuopio, Finland; University of Kuopio, 1988, pp 105-123.
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IP-32 © January 1990